Radikal Therapeutics (RTX) is developing a revolutionary bifunctional small molecule (R-801) for topical therapy of primary open angle glaucoma (POAG). R-801 is constructed from the covalent fusion of 2 chemical domains, each with demonstrated tissue protective properties: 1) a mito-K+-ATP channel activating moiety derived from the isoform non-selective K+-ATP channel opener pinacidil, and 2) a pyrrolidine nitroxide spin trap domain (hydroxymethylproxyl, HMP) that acts as superoxide dismutase (SOD) and catalase mimetics and a peroxynitrite decomposition catalyst. By design, R-801 targets 2 independent mechanisms of injury each considered critical to the pathogenesis of POAG: 1) intraocular hypertension (IOH), and 2) neuronal redox- mediated cell death. Our preliminary data reveal that R-801 reduces intraocular pressure (IOP) in explanted human eyes and in topically-treated mice. The present application is intended to extend these initial observations by elucidating: 1) the in vivo efficacy of the first mechanism of action (reduction in IOP) and toxicity in a clinically-relevant lapine model, and 2) the tissue distribution of R-801 in the relevant tissue compartments (aqueous humor, vitreous, trabecular meshwork, and retina) of the lapine eye. Specific Aim #1: Establish the dose-dependent toxicity and efficacy of topical R-801 ophthalmic solution. In Dr. Fautsch's laboratory, dose-response studies will be performed wherein 50 L of R-801 solution (0.002, 0.02, 0.2, 2.0% in buffered normal saline) will be applied to the bulbar conjunctivae of female Dutch Belted (pigmented) rabbits (n=6) for 7 days, followed by a 7 day washout period. IOP will be monitored TID by applanation tonometry during the treatment period and also 7 days pre and post treatment. Upon euthanasia, eyes will be harvested for detailed pathologic analysis for evidence of ocular toxicity, to be carried out by Dr J. Douglas Cameron. This Aim will provide in vivo data relating to the magnitude and duration of effect on IOP, the shape of the dose-response curve in regards to IOP reduction, and the ocular safety of topically-applied R-801 ophthalmic solution. Specific Aim #2: Establish the anterior chamber and retinal pharmacokinetics (PK) of topical R-801 ophthalmic solution. In conjunction with Dr. Fautsch, the aqueous humor, vitreous, trabecular meshwork, and retinas of female pigmented rabbits (n=4 per timepoint) will be harvested 2, 6, and 24 h after deposition of R-801 onto the bulbar conjunctiva (utilizing an optimal dose for IOP reduction defined in Aim #1). The concentration of R-801 in the retina, anterior chamber, and peripheral blood at each timepoint will be determined by LC-MS/MS. We expect that treatment with R-801 will satisfy the following criteria for progression to the Phase 2 SBIR stage: 1) Efficacy (> 35% statistically-significant reduction in IOP relative to vehicle control sustained for > 4 h), 2) Tissue uptake (aqueous chamber and retinal concentrations > 0.5% of formulation concentration), and 3) Safety (absence of local signs and symptoms of inflammation and irritation).